Cyclic adenosine monophosphate (cAMP) is a naturally occurring compound that is present in all cells and tissues, from bacteria to humans. In animal cells, cAMP appears to promote the expression of differentiated (specialized) properties. Well-known examples are the stimulation by cAMP of gluconeogenesis in liver, lipolysis in fat tissues, and water permeability in toad bladder epithelium. The functional development of the mammary gland is another example. The content of cAMP in rat mammary gland shows a diphasic pattern during the gestation cycle. The level of cAMP rises continuously toward the end of pregnancy, and then falls progressively to its lowest value by the 16th day of lactation. The transition at the time of parturition coincides with a considerable increase in the metabolic activity of the gland consequent to the onset of lactation.
Morphological alterations induced by cAMP in cultured cells in vitro include acinar formation and ultrastructural changes in thyroid cells, elongation and array formation in fibroblasts, development of neurite-like outgrowths in neuroblastoma cells, growth of processes in glioma cells, and pigmentation of melanocytes. The rapid changes in cell morphology are probably mediated through an effect by cAMP in interaction with Ca.sup.2+ on the cytoskeleton. Among the functional effects of cAMP are induction of specific enzymes stimulation of collagen synthesis in fibroblasts, and neurotransmitter synthesis in cells of neural origin. It is significant that cAMP-induced differentiation may occur without concomitant inhibition of cell division.
While a role for cAMP in cell differentiation seems to be established, it is much more difficult to define a general function for this nucleotide in the regulation of cell proliferation. The available data are interpreted very differently.
In fibroblasts, a number of studies have shown that treatment, which relieves growth inhibition in quiescent cells, such as serum addition or trypsinization, leads to decreased adenylate cyclase activity and a rapid fall in cAMP before the onset of DNA synthesis, supporting the idea that a drop in cAMP level is the decisive signal triggering cell division. However, conclusive evidence that the reduction in cAMP is causally related to the initial release from the resting state has not been presented. In cultured human fibroblasts, when DBcAMP or methylisobutylxanthine was added to prevent the cAMP drop during the first eight hours after serum addition, subsequent DNA synthesis was not delayed, whereas these agents were inhibitory when added more than eight hours after serum addition, in mid Gl, and in late Gl and S phases of the cell cycle. This indicates that a reduction in cAMP may not be involved as the initial trigger for serum-stimulated DNA synthesis, but it may be a factor at later stages, necessary for further progression toward DNA replication. However, in Balb/3T3 cells (Ca.sup.2+ deprived) stimulated by serum, there is an increase in cAMP in late Gl, suggesting a positive role for cAMP in the control of DNA replication in fibroblasts.
Apparent contradictory data also exist in the study of lymphocytes. Cyclic AMP in low concentrations (10.sup.-8 to 10.sup.-6 M) triggers DNA) synthesis in suspension cultures of rat thymic lymphocytes, providing one of the main arguments for a growth-promoting role of cAMP. Experiments on peripheral lymphocytes have yielded data that partly support and partly conflict with these results. For liver cells, there is increasing evidence that cAMP may have a positive effect on growth.
Studies showing growth inhibition by cAMP have tended to use higher concentrations of DBcAMP or the other agents than those studies showing growth stimulation, possibly indicating that the growth inhibitory effects seen are less physiological. It has become increasingly evident that effects seen after addition to biological systems of high concentrations of cAMP or DBcAMP are not necessarily direct effects of cAMP, but may be caused by metabolites, such as 5'AMP, adenosine, or butyrate. Similarly, methylxanthines have effects that probably are not mediated through cAMP. Physiologically, it seems that cAMP has several intracycle modulatory effects on normal cell growth, although it is quite certainly not the single growth regulator. Probably various growth factors operate by mechanisms independent of cAMP. In fact, cAMP may not be essential for cell cycle progression per se.
The effect of cAMP observed in malignant cells in culture in many cases constitutes a striking redifferentiation, which amounts to apparent renormalization of a number of properties of the transformed cells, including morphological features, adhesive properties, lectin agglutination, cell movement, biochemical functions, and anchorage-dependent growth.
It is unclear how fundamental this "normalization" is. For example, the tumorigenicity of DBcAMP-treated neuroblastoma cells has been reported to be decreased by Prasad (Biol. Rev. 50: 129-165, 1975), but was found to be unaltered in another study by Furmanski et al. (J. Schultz, and H. G. Gratzner, Eds., The Role of Cyclic Nucleotides in Carcinogenesis, pp. 239-261). New York and London: Academic Press, 1973. The presence of transformation-associated antigens on the cell surface is not prevented by cAMP. There is considerable variation in the morphological response to cAMP from cell to cell, even among fibroblasts.
Animal experiments have shown that various cAMP derivatives may inhibit: tumor growth in vivo (cf. Keller, Life Sci. 11: 485-491, 1972, and Cho-Chung et al., Science 183: 87-88, 1974). An interesting observation was that one single injection of cholera toxin, which is a potent adenylate cyclase activator, caused an almost complete inhibition of YAC lymphoma cell proliferation for up to four days in mice without noticeable toxic effects on the animals (Holmgren et al., Exp. Cell Res. 108: 31-39, 1977). Some of the most striking examples of malignant cell differentiation by cAMP in vitro have been observed in neuroblastoma cultures (Prasad, Biol. Rev. 50: 129-165, 1975).
A preliminary clinical study with the phosphodiesterase inhibitor papaverine, included in a combined drug regimen for disseminated neuroblastomas that in most cases were unresponsive to other drugs, has yielded promising results (Helson et al., J. Natl. Cancer Inst. 57: 727-729; 1976). Also, human oat cell lung carcinoma cells treated in vitro with DBcAMP have differentiated into neurone-like cells (Tsuji et al., Cancer Lett. 1: 311-318, 1976). Another interesting example is the induction of differentiation by DBcAMP of spindle cell sarcoma with multiple metastasis (Williams et al., Proc. Am. Assoc. Cancer Res. 23: 142, 1983). In this latter case, the patient was treated with DBcAMP (3-6 mg/kg) intravenously daily over five hours on days 1-9 and again on days 47-56. The tumor size plateaued and even decreased during both infusion periods and increased during intervals off treatment and after cessation of the second treatment. Histology of tumors biopsied on days 2, 14, and 60 showed evidence of differentiation during the DBcAMP infusion.
Because cAMP manifests almost ubiquitous biological effects, the unphysiologically high levels of cellular AMP that would result from prior compounds would disturb many cellular processes nonspecifically, resulting in a masking of a specific function of cAMP, such as growth regulation.
Cyclic AMP in mammalian cells functions via binding to its receptor protein, the regulatory subunit of cAMP-dependent protein kinase. There are at least two distinct isozymes for cAMP-dependent protein kinase, namely, type I and type II protein kinases, having different regulatory subunits but an identical catalytic subunit, and differential expression of these isozymes has been shown to be linked to regulation of cell growth and differentiation. Recently, two genes have been identified that code for two different catalytic subunits (C.alpha., C.beta.) of cAMP-dependent protein kinase. However, preferential coexpression of either one of these catalytic subunits with either the type I or the type II regulatory subunit has not been found.
Because a mixture of type I and type II kinase isozymes is present in most tissues, selective modulation of these isozymes in intact cells may be a crucial function of cAMP. All past studies of the cAMP regulation of cell growth employed either a few early known cAMP analogues that require an effective concentration of unphysiologically high millimolar range or agents that raise cellular cAMP to abnormally and continuously high levels. Under these experimental conditions, separate modulation of type I and type II kinase isozymes is not possible, because cAMP at high levels activates both isozymes maximally and equally without discrimination.
Recent studies on extensive cAMP binding kinetics, using purified preparations of protein kinase isozymes in vitro, identified site-selective cAMP analogs that selectively bind to either one of two different binding sites on the cAMP receptor protein. Furthermore, the site-selective analogues in appropriate combinations demonstrate synergism of binding and exhibit specificity toward either type I or type II protein kinase. This unique site-specificity of site-selective cAMP analogues is not mimicked by cAMP itself or by previously studied earlier analogues.
Human breast cancers often regress after hormone therapy, treatment that frequently involves the removal of the ovaries. The cancer regression has been linked to the presence of an ER in the tumor, to the extent that assays designed to measure the receptor are now used extensively to identify patients likely to respond to hormone therapy. However, it has become apparent that the mere presence of ER is not a reliable criterion for the response of mammary tumors to endocrine therapy. While patients with undetectable levels of tumor ER rarely respond to endocrine therapy, only 50-60% of ER-positive human breast tumors regress after hormone treatment. There is, therefore, a need to identify hormone-dependent cancers within the group of ER-positive tumors. The presence of PgR in ER-positive tumors has been reported to improve the prediction of endocrine responsiveness in some studies, but in other studies, PgR did not enhance prediction. The presence of PgR, therefore, does not necessarily improve the predictive value of ER. Additional discriminating factors are clearly required.
Evidence that cAMP receptor protein may represent such a parameter has come from studies in which regression of hormone-dependent mammary tumors followed administration of dibutyryl cyclic AMP, the effect being apparently mediated by cAMP receptor protein. Cyclic AMP receptor protein appears to be a marker of tumor sensitivity to hormonal manipulation as was shown in animal tumors and in a limited number of human breast cancers.
It has been suggested that regulation of the growth of hormone-dependent mammary tumors may depend on the antagonistic action between estrogen and cyclic AMP. Estrogen stimulates, whereas cAMP arrests, the growth of mammary carcinomas induced by 7,12-dimethyl-benz(.alpha.) anthracene (DMBA) in the rat. During growth arrest of the tumors, after either hormone removal via ovariectomy or treatment of the hosts with N.sup.6,O.sup.2' -dibutyryl-cAMP (DBcAMP), estrogen binding decreases; whereas cAMP binding and cAMP-dependent protein kinase activity increase in the cytosol and nuclei of the tumor cells. It was further demonstrated that the growth of DMBA-induced mammary tumors is associated with an enhanced expression of a cellular oncogene, c-ras. The p21 transforming protein of the ras gene product was a predominant in vitro translation product of mRNAs of the growing tumors, and a sharp reduction of the translated p21 protein preceded regression of these tumors after either ovariectomy or DBcAMP treatment.
Recent studies on cAMP binding kinetics, using purified preparations of cAMP-dependent protein kinases, identified cAMP analogues that are potent activators of protein kinase and selectively bind to either one of the two different cAMP binding sites of protein kinase (cf. Rannels et al. J. Biol. Chem. 255: 7085-7088, 1980). Generally, analogues modified at the C-8 position of the adenine ring are site 1-selective, and those modified at the C-6 position are site 2-selective. Furthermore, the Site 1- and Site 2-selective analogues in combination demonstrate synergism of binding to and activation of protein kinase, (Robinson-Steiner et al., J. Biol. Chem. 258: 1032-1040, 1983).